Alcohol abuse has long been known to impair lung host defenses increasing the risk in heavy drinkers for bronchitis, pneumonia and acute respiratory distress syndrome. Our laboratory has focused on alcohol's impact on mucociliary clearance in the conducting airways of the lung, which provides the first line of defense of the lung against inhaled infectious agents, particles and debris. We have established two key observations that drive this proposal: 1) brief modest alcohol intake stimulates airway ciliary motility; and 2) sustained heavy alcohol intake impairs mucociliary clearance increasing the risk for airway injury and pneumonia. We have termed this impairment Alcohol-Induced Ciliary Dysfunction (AICD). Our mechanistic studies have demonstrated that both the brief alcohol stimulation and sustained AICD involve ciliated cell mechanisms dependent on the regulation of nitric oxide (NO), cilia-associated cyclases and cyclic nucleotide-dependent kinases (PKG & PKA). Recently, we published that these alcohol-triggered ciliary regulation pathways are distinctly localized to the basal body of each airway cilium in what we have called the alcohol-responsive ciliary metabolon. This pathway is exquisitely sensitive to very low concentrations of alcohol (1-10 mM, which is less than half of the legal intoxication limit) and is fully functionl in isolated airway cilia organelles. This leads us to hypothesize that: Alcohol causes reversible airway ciliary dysfunction due to modified nitric oxide signaling and altered regulation of key cila proteins, including the dynein motors and the mechanisms that regulate dyneins. In this proposal, we outline experiments designed to answer four new questions: 1) How does brief alcohol exposure stimulate NO production in airway cilia? We explore this question in Aim #1 by examining the role alcohol plays in activating nitric oxide synthase (eNOS) by enhancing the chaperone function of eNOS by heat shock protein 90 (HSP90). 2) Why does sustained alcohol exposure deplete ciliary NO levels? We address this question in Aim #2 by defining how eNOS becomes uncoupled by sustained alcohol exposure due to L- arginine depletion and the generation of reactive oxygen species (ROS). We think increased ciliary ROS leads to depletion of the eNOS cofactor tetrahydrobiopterin (BH4) resulting in cilia desensitization. 3) How do cilia regulatory enzymes interact to modify motility? In Aim #3 we will determine how sustained alcohol exposure activates protein phosphatase 1 (PP1), which leads to dephosphorylation of HSP90 and other key cilia activation proteins such as PKA. 4) What downstream cilia motor molecules are modified by alcohol exposure? Using the model genetic system of the alcohol-sensitive motile organism, Chlamydomonas, Aim #4 will focus on how sustained alcohol modifies ciliary outer dynein arms, which are the motor proteins that make cilia beat. The studies we propose will greatly extend our knowledge of how to prevent and treat AICD and will expand our insight into how alcohol alters cilia molecules critical for airway cilia function. 1